This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
RHOT2 / Mitochondrial Rho GTPase 2 · IHC design guide
Design Immunohistochemistry for RHOT2
Plan RHOT2 paraffin-section IHC around cytoplasmic staining in most tissues (HPA tissue IHC). Start the catalog antibody at 2–5 μg/ml (datasheet A08801-1), using high-staining glandular cells and adipocytes reported as undetected to assess the result (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RHOT2 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic tissue staining (HPA tissue IHC); outer mitochondrial membrane (UniProt) | |
| Staining pattern | Cytoplasmic staining in most tissues; high in glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08801-1) | |
| Positive control | Cerebral cortex+4 more · see all | |
| Negative control | Adipose tissue+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium consistency with RNA data (HPA tissue IHC) | |
| Regulation | Ubiquitous expression (UniProt) | |
| Isoform / epitope | 2 isoforms; antibody epitope coverage is unreported (UniProt) |
Section 1
Recommended RHOT2 IHC & IF Protocols
The catalog antibody protocol (datasheet A08801-1) and one published FFPE tumor protocol (PMC10234629) provide starting conditions for RHOT2 IHC.
| Sample | Paraffin-embedded human prostate cancer tissue; fixative not specified (datasheet A08801-1) |
| Fixation | Image fixative and duration unreported (datasheet A08801-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A08801-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08801-1) |
| Primary antibody | Rabbit anti-RHOT2, 2-5 μg/ml (datasheet A08801-1) |
| Primary incubation | Overnight at 4 °C (datasheet A08801-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A08801-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RHOT2-positive staining in neuronal cells of cerebral cortex (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A08801-1); the published tumor protocol does not report retrieval conditions (PMC10234629).
Section 2
What Is the Expected RHOT2 Staining Pattern?
RHOT2 is an outer mitochondrial membrane protein with residues 1–592 facing the cytoplasm (UniProt Q8IXI1 topology). In paraffin tissue sections, expect cytoplasmic staining in many cell types, including strong staining in selected neurons, glandular cells, trophoblasts and myocytes (HPA tissue IHC: High in those cells). HPA rates its tissue staining Approved, with medium agreement between staining and RNA data; external verification is pending (HPA tissue IHC reliability).
| Cytoplasmic staining is strong in cerebral cortex neurons, intestinal glandular cells or skeletal myocytes (HPA tissue IHC: High). | This fits the reported cell and tissue pattern. Assess staining within the relevant cells rather than treating every cell in a positive section as equally positive (HPA tissue IHC: cell-specific levels). |
| Signal is predominantly nuclear, or confined to a compartment inconsistent with cytoplasmic staining (HPA tissue IHC: cytoplasmic expression). | Treat the compartment mismatch as suspect and inspect controls and staining conditions. RHOT2 resides on the mitochondrial outer membrane; a nuclear-only pattern does not match that location (UniProt Q8IXI1 subcellular location). |
| Adipocytes or peripheral nerve show conspicuous staining where HPA reports no detection (HPA tissue IHC: Not detected). | Consider antibody cross-reactivity or endogenous detection activity, then compare with controls. HPA's negative observations are useful comparators, but do not alone identify the cause of an unexpected signal (HPA tissue IHC: Not detected). |
| Broad color deposit obscures cell boundaries or appears outside identifiable cells. | Interpret the slide cautiously: nonspecific background can hide a true cytoplasmic pattern. Check the detection-only control, blocking, wash steps and chromogen development before scoring cells (general IHC practice). |
| No signal appears in colon glandular cells or cerebral cortex neurons (HPA tissue IHC: High). | A failed positive control is possible. Review tissue integrity, retrieval, primary antibody and detection controls before calling RHOT2 absent; an unstained section cannot overturn the reported tissue pattern (general IHC practice; HPA tissue IHC: High). |
💡Expected RHOT2 appearanceCall a positive result when cytoplasmic signal is strong in HPA-high cells such as colon glandular cells or skeletal myocytes; predominant nuclear staining or prominent signal in HPA-negative adipocytes is suspect (HPA tissue IHC: High, Not detected, cytoplasmic expression; UniProt Q8IXI1 location).
| Compartment and topology | RHOT2 is anchored in the mitochondrial outer membrane by a segment at residues 593–615, with most of the protein facing the cytoplasm (UniProt Q8IXI1 topology). In chromogenic IHC, assess the reported cytoplasmic pattern; the record does not specify how sharply individual mitochondria resolve (HPA tissue IHC: cytoplasmic expression). |
| Choice of tissue comparator | HPA reports High staining in colon, duodenum, rectum, small intestine and stomach glandular cells, among other listed cells; adipocytes and peripheral nerve are Not detected (HPA tissue IHC). Choose a positive cell population that can be identified on the section, and interpret negative comparators at the cell level (general IHC practice). |
| Evidence and antibody validation | The tissue profile is Approved, with medium staining-to-RNA consistency and external verification pending (HPA tissue IHC reliability). HPA012624 and HPA012895 are IHC Approved, while only HPA012624 is ICC Supported in the supplied antibody record (HPA antibodies). Approval supports use of the observed pattern as a comparator, with the stated verification limit (HPA tissue IHC reliability). |
| IF/ICC Q&A: Where should RHOT2 appear? | In ICC-IF, HPA reports supported mitochondrial localization, with images from HEK293, SH-SY5Y and U2OS (HPA subcellular ICC-IF). That localization is consistent with the outer membrane assignment (UniProt Q8IXI1 location). This answers the localization question; the supplied record gives no IF/ICC protocol option. |
| Isoforms and epitope information | UniProt lists 2 isoforms and a full-length chain spanning residues 1–618 (UniProt Q8IXI1 isoforms and processing). The supplied record does not map either antibody's epitope, so it cannot establish isoform-specific staining or predict an antigen-retrieval response. |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported high-staining cell population is blank (HPA tissue IHC: High). | The run or detection system may have failed; absence in one section does not establish absent protein (general IHC practice). | Check a known-positive tissue in the same run, reagent steps and detection control. Reassess the sample only after the positive control stains as expected (general IHC practice; HPA tissue IHC: High). |
| Signal is nuclear rather than cytoplasmic (HPA tissue IHC: cytoplasmic expression). | The distribution conflicts with the reported mitochondrial outer membrane location (UniProt Q8IXI1 location). | Compare with a positive tissue and appropriate negative controls; withhold a positive call if the compartment mismatch persists (general IHC practice; HPA tissue IHC: cytoplasmic expression). |
| Adipocytes stain strongly despite a Not detected HPA observation (HPA tissue IHC: adipocytes). | Cross-reactivity or detection background is possible; the HPA observation alone cannot distinguish them (HPA tissue IHC: Not detected; general IHC practice). | Inspect a detection-only control and compare staining in neighboring identifiable cells before interpreting the adipocyte signal (general IHC practice). |
| Diffuse chromogen covers both tissue and empty areas. | Background from detection, washing or chromogen development may obscure cell-specific staining (general IHC practice). | Review blocking, washes and development time; compare with a detection-only control before scoring the section (general IHC practice). |
| The chosen positive tissue has weak staining in the cells being scored. | HPA levels differ by cell type: pancreatic endocrine cells and thyroid glandular cells are Low, whereas colon glandular cells are High (HPA tissue IHC). | Identify the cell population first and compare it with its reported level; use a listed High cell population to assess run performance (HPA tissue IHC; general IHC practice). |
| A retrieval change produces an unexpected staining difference. | The supplied sources give no RHOT2-specific fixation or antigen-retrieval sensitivity, so the difference has no supported target-specific explanation. | Compare conditions on matched sections with the same positive and detection controls, then document the observed effect without assigning a RHOT2-specific mechanism (general IHC practice). |
Experiment evidence
IHC & IF images showing RHOT2 staining
A08801-1 · IHCIHC analysis of MIRO2/RHOT2 using anti-MIRO2/RHOT2 antibody (A08801-1). MIRO2/RHOT2 was detected in a paraffin-embedded section of human pro
A08801-1 · IHCIHC analysis of MIRO2/RHOT2 using anti-MIRO2/RHOT2 antibody (A08801-1). MIRO2/RHOT2 was detected in a paraffin-embedded section of human cer
A08801-1 · IFIF analysis of MIRO2/RHOT2 using anti-MIRO2/RHOT2 antibody (A08801-1) and anti-Tubulin Alpha antibody (M03989-3). MIRO2/RHOT2 was detected i
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000140983-RHOT2/tissue/cerebral+cortex#imid_3359288
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000140983-RHOT2/tissue/colon#imid_3359206
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000140983-RHOT2/tissue/liver#imid_3359207
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000140983-RHOT2/tissue/kidney#imid_3359208
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA012624; green = RHOT2, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA012624; green = RHOT2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RHOT2 IHC & IF
🧪Run colon first and require staining in glandular cells (HPA: High in colon glandular cells); use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes). On the colon slide, use gland lumina and non-glandular areas to assess background, without assuming that non-glandular cells are RHOT2-negative (HPA: glandular-cell result only).
Positive control tissue: Cerebral cortex (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RHOT2 in HEK293, SH-SY5Y, U2OS, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and matched rabbit IgG isotype controls (A08801-1 caption: rabbit primary and goat anti-rabbit secondary), plus a RHOT2 knockout specimen processed alongside the section as a biological negative (standard IHC practice). For colon HRP/DAB staining, quench endogenous peroxidase and inspect gland lumina for nonspecific chromogen deposits (A08801-1 caption: HRP/DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A08801-1 paraffin-section caption does not state a fixative (A08801-1 caption). Heat-mediated EDTA retrieval at pH 8.0 is a documented IHC starting condition, but retrieval dependence has not been established (A08801-1 caption). Frozen-section feasibility is unreported; IF/ICC offers a way to assess mitochondrial localization (HPA: supported mitochondrial location), but the supplied evidence does not establish which application is easier, and luminal DAB deposits can complicate colon scoring (standard IHC practice).
HPA tissue IHC evidence for RHOT2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced RHOT2 IHC Tips
Troubleshoot RHOT2 staining in paraffin sections by checking retrieval, compartment, controls, and scoring before interpreting signal.
Which retrieval condition should I start with for RHOT2 paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A08801-1). That condition precedes RHOT2 detection in the catalog antibody’s paraffin-section image, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A08801-1). If staining is weak, compare a modest range of heating times on adjacent sections while keeping the buffer and primary concentration fixed, and inspect tissue integrity after cooling (standard IHC practice). Include a consistently stained reference section and a no-primary control in each comparison so increased DAB deposition is not mistaken for improved antigen exposure (standard IHC practice).
Does fixation explain weak RHOT2 staining in my paraffin sections?
Target-specific RHOT2 sensitivity to fixation is unknown: the selected paraffin-section caption does not report a fixative (datasheet A08801-1). Record the fixative, fixation duration, section age, and processing history for each specimen before comparing staining intensity (standard IHC practice). Compare sections processed alike using EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody overnight at 4°C, the conditions reported for the catalog image (datasheet A08801-1). If processing histories differ, test matched reference tissue alongside the specimens and treat intensity differences cautiously until handling effects have been separated from biological variation (standard IHC practice).
What RHOT2 staining pattern is plausible in chromogenic IHC?
RHOT2 is assigned to the mitochondrial outer membrane, with residues 1–592 facing the cytoplasm and a membrane segment at 593–615 (UniProt Q8IXI1 topology). Its mitochondrial location is supported by subcellular imaging, while tissue IHC reports cytoplasmic expression in most tissues (HPA subcellular; HPA tissue IHC). In a chromogenic section, assess granular cytoplasmic signal in intact cells against the surrounding DAB background; light microscopy may not resolve individual mitochondria (standard IHC practice). Diffuse staining alone cannot establish mitochondrial localisation, so compare morphology and controls before assigning the signal to RHOT2 (standard IHC practice).
Could isoforms or epitope position change my RHOT2 IHC result?
RHOT2 has 2 listed isoforms, but the supplied antibody caption does not identify its epitope or establish isoform coverage (UniProt Q8IXI1 isoforms; datasheet A08801-1). The protein includes Miro regions at residues 2–168 and 414–576, two EF-hands, and a C-terminal membrane segment at 593–615 (UniProt Q8IXI1 domains and topology). Check the antibody’s documented immunogen or epitope before attributing a negative section to loss of a particular isoform (standard IHC practice). If that information is unavailable, report staining as antibody-detected RHOT2 and avoid isoform-specific claims, even when a control section stains as expected (standard IHC practice).
How should I follow up RHOT2 IHC with multiplex immunofluorescence?
Use IF as a separate assay and pair RHOT2 with a marker for the expected cell population and a mitochondrial marker to assess cellular identity and compartment together (standard IF practice; HPA subcellular). Choose spectrally separated fluorophores, placing a brighter, longer-wavelength channel where tissue autofluorescence is troublesome, and check single-stain controls for bleed-through (standard IF practice). RHOT2 residues 1–592 face the cytoplasm, whereas residues 616–618 face the mitochondrial intermembrane space (UniProt Q8IXI1 topology). Set permeabilisation according to the documented antibody epitope and fixation method; without epitope information, compare mild permeabilisation conditions rather than assuming access across the outer membrane (standard IF practice).
How can I distinguish RHOT2 signal from chromogenic background?
First inspect the no-primary control and any signal in acellular areas, vessel contents, or damaged tissue before changing the RHOT2 antibody concentration (standard IHC practice). The catalog image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary, and DAB development (datasheet A08801-1). Include a peroxidase-blocking step and control DAB development time when using this detection chemistry (standard IHC practice). If background persists, compare adjacent sections with shorter chromogen exposure and verify that the secondary alone does not reproduce the cellular pattern (standard IHC practice).
How should I score RHOT2 across differently composed tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: RHOT2 is assigned to mitochondria, and tissue IHC describes cytoplasmic expression in most tissues (UniProt Q8IXI1 subcellular; HPA tissue IHC). Within the same cell type, record an H-score from percentage of cells at each intensity, or report percentage positive using a prespecified threshold (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable, evaluable tissue and exclude folds, edges, and necrosis (standard IHC practice). Normalise to the relevant cell count or tissue area, then compare matched processing batches and a common reference section before interpreting small differences (standard IHC practice).
What would make a positive RHOT2 IHC result convincing?
Look for reproducible cytoplasmic signal in intact cells, compatible with RHOT2’s mitochondrial outer-membrane assignment and the reported tissue IHC pattern (UniProt Q8IXI1 subcellular; HPA tissue IHC). High staining has been reported in skeletal-muscle myocytes and several glandular cell populations, while adipocytes were reported as not detected; these observations are reference patterns, not universal pass/fail controls (HPA tissue IHC). Treat isolated nuclear signal, staining confined to section edges or necrosis, and signal reproduced without primary antibody as reasons to investigate artefact (standard IHC practice). Check peroxidase blocking when unexplained DAB deposition persists, and avoid identifying RHOT2 solely from brown colour without cell and compartment context (standard IHC practice).
Boster reagents
Best RHOT2 / Mitochondrial Rho GTPase 2 IHC Antibodies
Anti-RHOT2 antibody A08801-1 has IHC images from human prostate, cervical and renal cancers and mouse stomach, plus IF/ICC data in U2OS cells (catalog image captions).
Real IHC data
Anti-MIRO2/RHOT2 Antibody ®
Cat # A08801-1
A08801-1 is shown in paraffin-section IHC images of human prostate, cervical and renal cancers and mouse stomach (A08801-1 IHC image captions). It is also shown in IF/ICC imaging of U2OS cells (A08801-1 IF image caption).
Which to pick: Choose A08801-1 for paraffin-section tissue IHC: its own captions document staining in human cancer tissues and mouse stomach; the fixative is unreported (A08801-1 IHC image captions). Choose the same SKU for IF/ICC because its application list includes both and its IF caption shows U2OS cells (A08801-1 catalog and IF image caption). For cross-species work, its listed reactivity is Human, Mouse and Rat, while the supplied IHC images document Human and Mouse only (A08801-1 catalog and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8IXI1 (MIRO2_HUMAN, Mitochondrial Rho GTPase 2).
- Human Protein Atlas. RHOT2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RHOT2 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. RHOT2 antibody validation summary (2 antibodies).
- Proteomic characteristics reveal the signatures and the risks of T1 colorectal cancer metastasis to lymph nodes. eLife 2023 — PMC10234629.
- Identification of Prognostic and Metastatic Alternative Splicing Signatures in Kidney Renal Clear Cell Carcinoma. Frontiers in bioengineering and biotechnology 2019 — PMC6803439.
- PubMed PMID:12482879 — UniProt-cited evidence.
- PubMed PMID:15218247 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RHOT2 (Mitochondrial Rho GTPase 2). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); outer mitochondrial membrane (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08801-1). Validated IHC antibody: A08801-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebral cortex (Neuronal cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human prostate cancer tissue; fixative not specified (datasheet A08801-1); 2. Fixation: Image fixative and duration unreported (datasheet A08801-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A08801-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08801-1); 8. Primary antibody: Rabbit anti-RHOT2, 2-5 μg/ml (datasheet A08801-1); 9. Primary incubation: Overnight at 4 °C (datasheet A08801-1). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Staining has medium consistency with RNA data (HPA tissue IHC); Isoform / epitope: 2 isoforms; antibody epitope coverage is unreported (UniProt); Regulation: Ubiquitous expression (UniProt). Evidence: UniProt Q8IXI1, HPA tissue IHC · Approved, HPA ICC-IF · mitochondria., Datasheet A08801-1, PMC10234629. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RHOT2/immunohistochemistry-ihc-guide.